Acta Univ Latv (2008) 745: 145–154

Specificity of packaging mRNAs in bacteriophage GA virus-like particles in yeast Saccharomyces cerevisiae

Abstract

Heterologous expression of RNA bacteriophage coat protein genes leads to formation of viruslike particles that encapsulate intracellular RNA. Here we packaged specific mRNA molecules into bacteriophage GA capsids in vivo. For specificity we employed a GA operator – a 21-nucleotide-long RNA sequence that folds into a stem-loop structure and can specifically bind to the coat protein of the phage. Several plasmids were constructed for expression in Saccharomyces cerevisiae, which directed synthesis of both the GA coat protein and the mRNA to be packaged. As models for packaging, three different mRNAs (GA coat protein, ENA-78 and GFP) were used, each constructed in two versions that differed by the presence of the GA operator within their sequence. RNA content analysis of the produced capsids in gel electrophoresis revealed the existence of RNAs of predicted lengths. The presence of the packaged mRNAs in the capsids was further verified by reverse transcription PCR. However, the operator had rather small effect on the specificity of capsid contents, since the mRNA of GA coat protein was also invariably detected inside the capsids.